PT2385 is a first-in-class, selective small-molecule inhibitor of hypoxia-inducible factor-2alpha (HIF-2alpha) developed for the treatment of advanced clear cell renal cell carcinoma. Preclinical results demonstrated that PT2385 has potent antitumor efficacy in mouse xenograft models of kidney cancer. Recent studies revealed that PT2385 inhibits in the intestine and has efficacy in the treatment of diet induced obesity, insulin resistance and fatty liver disease. It also has activity toward metabolic disease in a mouse model. However, no metabolism data are currently publicly available. It is of great importance to characterize the metabolism of PT2385 and identify its effect on systemic homeostasis in mice. High-resolution mass spectrometry-based metabolomics was performed to profile the biotransformation of PT2385 and PT2385-induced changes in endogenous metabolites. Liver microsomes and recombinant drug-metabolizing enzymes were used to determine the mechanism of PT2385 metabolism. Real-time polymerase chain reaction analysis was employed to investigate the reason for the PT2385-induced bile acid dysregulation. A total of 12 metabolites of PT2385 was characterized, generated from hydroxylation (M1, M2), dihydroxylation and desaturation (M3, M4), oxidative-defluorination (M7), glucuronidation (M8), N-acetylcysteine conjugation (M9), and secondary methylation (M5, M6) and glucuronidation (M10, M11, and M12). CYP2C19 was the major contributor to the formation of M1, M2, and M7, UGT2B17 to M8, and UGT1A1/3 to M10-M12. The bile acid metabolites taurocholic acid and tauro-beta-muricholic acid were elevated in serum and liver of mice after PT2385 treatment. Gene expression analysis further revealed that intestinal HIF-2alpha inhibition by PT2385 treatment upregulated the hepatic expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis. This study provides metabolic data and an important reference basis for the safety evaluation and rational clinical application of PT2385. Rutaecarpine (RUT), evodiamine (EOD) and dehydroevodiamine (DHED), are the three main bioactive indoloquinazoline alkaloids isolated from Euodia ruticarpa, a widely prescribed traditional Chinese medicine. These compounds have a variety of intriguing biological properties such as antitumor, antithrombotic, anti-inflammatory, analgesic, anti-obesity, anti-cholinesterase and anti-amnesic activities. The structure-activity relationships of these analogues for aryl hydrocarbon receptor (AHR) activation were explored by use of Ahr-deficient (Ahr-/-) mice, primary hepatocyte cultures, luciferase reporter gene assays, in silico ligand docking studies and metabolomics. In vitro, both mRNA analysis of AHR target genes in mouse primary hepatocytes and luciferase reporter assays in hepatocarcinoma cell lines demonstrated that RUT, EOD and DHED significantly activated AHR, with an efficacy order of RUT DHED EOD. Ligand-docking analysis predicted that the methyl substitute at the N-14 atom was a key factor affecting AHR activation. In vivo, EOD was poorly orally absorbed and failed to activate AHR, while RUT and DHED markedly upregulated the hepatic AHR battery gene expression in wild-type mice, but not in Ahr-/- mice. Furthermore, RUT, EOD and DHED were not hepatoxic at the doses employed. However, RUT and DHED disrupted bile acid homeostasis in an AHR-dependent manner. These findings revealed that the methyl group at the N-14 atom of these analogues and their pharmacokinetic behaviors were the main determinants for AHR activation. These data suggest that attention should be given to monitoring bile acid metabolism in the clinical use of Euodia ruticarpa. Triptolide, a major active constitute of Tripterygium wilfordii Hook. F, is prescribed for the treatment of autoimmune diseases in China. One of its most severe adverse effects observed in the clinical use is hepatotoxicity, but the mechanism is still unknown. Therefore, the present study applied an LC/MS-based metabolomic analysis to characterize the metabolomic changed in serum and liver induced by triptolide in mice. Mice were administered triptolide by gavage to establish the acute liver injury model, and serum biochemical and liver histological analysis were applied to assess the degree of toxicity. Multivariate data analyses were performed to investigate the metabolic alterations. Potential metabolites were identified using variable importance in the projection values and student's t-test. A total of 30 metabolites were observed that were significantly changed by triptolide treatment and the abundance of 29 metabolites were correlated with the severity of toxicity. Pathway analysis indicated that the mechanism of triptolide-induced hepatotoxicity was related to alterations in multiple metabolic pathways, including glutathione metabolism, tricarboxylic acid cycle, purine metabolism, glycerophospholipid metabolism, taurine and hypotaurine metabolism, pantothenate and CoA biosynthesis, pyrimidine metabolism and amino acids metabolism. The current study provides new mechanistic insights into the metabolic alterations that lead to triptolide-induced hepatotoxicity.